Onion is preferred as a commercial vegetable crop all over the
world, since it ranks second in value in the list of cultivated
vegetable crops. Area under its cultivation is 3.44 million hectares and
its total annual production in the world is 61.6 million tonnes (FAO,
2006).

The most important qualitative genes in the edible alliums are
those that cause male sterility. In onion, male sterility was first
exploited by Jones and Clarke (1943) using a male sterile specimen of
cultivar Italian Red, which they found in breeding plots at Davis,
California, in 1925. Fortunately, when this plant was prevented from
being cross pollinated, bulbils were produced in the flower head and it
could be propagated. Jones and Clarke (1943) published this classical
work describing the genetics of male sterility and indicating how it
could be used to produce hybrid cultivars. On the basis of this
technique, originally developed in onion, male sterility has since been
exploited in hybrid breeding of more than 150 crop species (Kale and
Munjal, 2005).

The genetic basis of the cytoplasmic male sterility (CMS) in
Italian Red is simple: a sterility inducing cytoplasm (S) and a nuclear
restorer locus with two alleles, Ms and ms. Male fertility is restored
by the dominant allele. The fertility restorer genes were later found
extensively in onions collected from various countries (Davis, 1957).

In the 1960s, another CMS cytoplasm, namely T-cytoplasm, was
discovered (Berninger, 1965; Schweisguth, 1973). This source of male
sterility was found in the French cultivar Jaune Paille de Vertus. The
T-cytoplasm is restored by two independently operating restorer systems.
The first restorer system comprised of a single locus, A, with two
alleles, in which male fertility is restored by the dominant allele. The
second restorer system is comprised of two loci, B and C, with
complementary gene action. Male fertility in this restorer system is
restored only when a dominant allele is present on both B and C loci.
Meer van der and Van bennekom (1969) observed thermolability, when
analyzing a CMS line, which originated from Polish cultivar Wolska,
which is a source of T-cytoplasm. They found 93% male sterile plants at
14 0C and only 10% at 23 0C. This complex fertility restoration has
resulted in negligible use of T-cytoplasm for hybrid development. The
majority of hybrid onion cultivars produced upto date are using S
cytoplasm (Havey, 1995; 2000) as a source of male sterility. This source
of male sterile cytoplasm traces back to a single onion plant identified
in Davis, California, in 1925 (Jones and Emsweller, 1936). Cytoplasmic
male sterility is found to have ascribed to mitochondrial genomes. Many
scientists (Courcel et al., 1989; Holford et al., 1991; Havey 1993,
1995; Satoh et al., 1993; Sato, 1998) have identified molecular markers
that distinguish normal (N) male-fertile and (S and T) sterile
cytoplasms of onions.

In 1970, T-cytoplasm carrying maize hybrids suffered heavy losses
due to epidemic of southern corn leaf blight (Pring and Lonsdale, 1989,
Levings, 1993). This single incidence has forced plant scientists to
think seriously about cytoplasmic uniformity and genetic vulnerability
of crop plants (Anonymous,1972).So far, only one type of male sterile
cytoplasm (CMS S) has been found useful. Newer types of male sterility
needs to be studied which will reduce genetic vulnerability of onions
and also useful for commercial production of bulb and seed onions at
lesser costs (Gokce et al., 2002).

In our study, we have screened onion population for occurrence of
male sterile plant/s, tissue cultured them, studied using molecular
markers and by making crosses with other onion varieties.

Materials and Methods

All of the genotypes mentioned in Table 1 were planted in the field
during Rabi 2003. After harvesting, the bulbs of these varieties were
preserved in cold storage for six months at 40C and 65 % relative
humidity. Bulbs were taken out during 1st week of November. Planting of
all these bulbs was done in Rabi 2004. All the genotypes bolted and
flowered in January 2005. Umbels of these genotypes were checked
physically and by acetocarmine treatment for assessing their male
sterility.

Genomic DNA was extracted from all the genotypes mentioned in Table
1. A systematic study of cytoplasms of all the genotypes was undertaken
using polymerase chain reaction (PCR) and primers as described by Sato
(1998) and Engelke and Tatlioglu (2002). The PCR-marker which anchors in
the upstream region to the mitochondrial gene cob is referred to as
5' cob-marker (Sato, 1998). These markers are able to distinguish
between S and N cytoplasm. T cytoplasm which was not studied by
Sato(1998) shows the same amplified product as N cytoplasm. Therefore,
Engelke and Tatlioglu (2002) developed a new orfA501-marker that
amplifies in S and T cytoplasm of onion, but not in N cytoplasm. The
combination of the 5' cob-marker with the orfA501-marker allows to
distinguish between these three cytoplasm types in individual plants.

Markers used for classification of cytoplasms are tabulated in
Table 2.

PCR reaction mixture used for amplification is shown in Table 3.

Table 3: PCR reaction mixture used for amplification PCR was
carried out using Mastercycler gradient from Eppendorf, using
amplification conditions (Table 4).

The crosses were made involving suspected male sterile plant as
female parent and other varieties (Arka Kirtiman,Arka Pitambar, Udaipur
102, Pusa White Round, Pusa White Flat, Punjab White, Gujarat Local,
Phule Safed) as male parent. All these crosses were made in Rabi 2005
and seeds of these crosses were planted in Rabi 2006. Bulbs of these
crosses were stored in cold storage until planting in Rabi 2007.

Results and Discussion

Field screening

All the onion genotypes were screened for presence of male
sterility. Results of screening are summarized in Table 1.

All the genotypes have shown male fertility, except V7 and Indam,
while JISL5 has shown segregation for male sterility. While screening
these genotypes for male sterility, we have got one plant in Agrifound
white population which was showing male sterility. We have tissue
cultured it and planted 100 plantlets in late kharif 2005. All of these
plants flowered subsequently and found out to be male sterile.
Subsequently, these plants have been again planted in the next season
i.e. late kharif 2006 and confirmed their male sterility.

Molecular studies

Results of PCR based analyses of cytoplasm are given in Table 5.

Table 5: PCR based analyses of cytoplasm

Results of Table 5 show that genotypes V7, Arka Kirtiman and Arka
Pitambar and Indam possess S cytoplasm, while rest of the genotypes
possess N cytoplasm. JISL5 is the only genotype with T cytoplasm.
Agrifound White A was showing male sterility in the field, but to our
great surprise its cytoplasm was found to be male fertile.

Genotype Agrifound White A was showing male sterility in the field,
while its PCR based molecular character indicated it to have N type
cytoplasm. In order to understand the nature of male sterility, crosses
have been made involving Agrifound White A as female parent and other
varieties (Arka Kirtiman,Arka Pitambar, Udaipur 102, Pusa White Round,
Pusa White Flat, Punjab White, Gujarat Local, Phule Safed) as male
parent. All these crosses were made in Rabi 2005 and seeds of these
crosses were planted in Rabi 2006. Bulbs of these crosses were preserved
in cold storage until planting in Rabi 2007. Results of these crosses
are presented in Table 6.

Crosses involving Agrifound White A as female parent were made in
order to study the nature and dominance of male sterility. Results of
these crosses in Table 6 show male fertility of these hybrids.
Interestingly, all the crosses irrespective of their cytoplasm and
nuclear background, restored fertility in Agrifound White A. Analysis of
sterility observed in Agrifound White A indicated that it is probably of
GMS ( Genetic male sterility) type and the male fertility is dominant
over male sterility and is a new source of male sterility in onion.
Genetic male sterility is of very wide occurrence in flowering plants
and as many as 60 genes for maize, 55 in tomato, 10 in cotton and 60 in
rice are known (Horner and Palmer, 1995).